The Role Of MALAT1/miR-329-5p/PRIP1 Signaling And Biomechanics In Steroid-associated Osteonecrosis Of Femoral Head

[Objective]In order to solve the challenge of treatment of patients with steroid-induced osteonecrosis of femoral head(SIOFH),we established a rat model of steroid-induced osteonecrosis of femoral head and gave treadmill training and tail suspension test to search the mechanism of biomechanical stimulation in SIOFH.After the rats were sacrificed,both sides of the femoral head tissue were obtained.We analyses the relative expression of MALAT1 / miR-329-5p / PLDL1 signaling pathway by molecular biology in the rat femoral head.In vitro,we induced cell lines(RAW264.7 and MC3TE1)to differentiate to osteoclast and osteoblast,and then gave siRNA transfection after dexamethasone intervention to further validate the metabolism of MALAT1 / miR-329-5p / PLDL1 signaling pathway in steroid-induced osteonecrosis of femoral head.[Method]Part one: the total of thirty eight-week-old SD rats were randomly distributed to control group(five)and SIOFH group(twenty-five),which were injected methylprednisolone sodium succinate by daily intramuscular.From the beginning of fourth week,we did the MRI scan every two weeks to observe the change of femoral head.After eight weeks when the process was finished,we randomly selected ten rats from SIOFH group,and gave them tail suspension test for three or six weeks.We selected another ten rats from SIOFH group,and then gave treadmill training for two or four weeks.Bilateral femoral head were obtained after the rats were sacrificed,and one of femoral head was frozen in a refrigerator at-80°C,the other was was soaked in 4% paraformaldehyde for Micro-CT scanning.The femoral head was decalcified with 10% EDTA after Micro-CT scanning and used for H-E staining.Part two: miRNAs and Lnc RNAs related to phospholipase C-related catalytically inactive proteins were searched by Mouse Genome Informatics and literature,and we used real-time PCR and Western blotting to analyses Lnc RNAs,miRNA,mRNA,and target protein expression levels in the femoral head tissue of the blank control group,SIOFH group,rat tail suspension group and treadmill training group,and to further study the mechanism of biomechanics in steroid-induced osteonecrosis femoral head.Part three: In vitro experiments,RAW264.7 and MC3T3E1 could be induced into osteoclasts and osteoblasts,giving dexamethasone intervention could simulate and observe the effect of glucocorticoid on bone metabolism abnormalities in steroid-induced osteonecrosis femoral head.The mechanism of Lnc RNA/miRNA/PLDL1 signaling pathway on the formation of osteoclasts and osteoblasts in steroid-induced osteonecrosis femoral head was analyzed by RT-PCR and Western blotting after lipo2000 transfection into siRNA.[Results]Part one:Through the observation the microstructural of femoral head in Micro-CT scans images and HE staining pictures of the blank control group,steroid-induced osteonecrosis of femoral head group,tail suspension test(TST)group and treadmill training group,we found that bone mineral density(BMD)decreased significantly in SIOFH group compared with blank control group,and gaven treadmill training,the bone mineral density has increased.However,after treadmill 4 weeks,the trabecular structure of normal arrangement disappear,appear messy,even dense cortical bone structure in weight-bearing area,and don't appear new trabecular structure in non-weight-bearing area;in tail suspension group,the bone density decreased slightly and the bone trabecula became narrowed,but the structure of the trabecula tended to become normal,we used Inveon Aanlysis soft to quantitative analyses the change of trabeculand bone marrow,its results is coincident with the observed results and further verified our conclusion.So we found that abnormal bone metabolism plays a vital role in the steroid-induced osteonecrosis of femoral head and biomechanics on SIOFH.Part II: Real-time fluorescence quantitative PCR and Western Blot results show that PLDL1 is involved in the occurrence of steroid-induced femoral head necrosis at the mRNA level and protein level.Increased expression level of PLDL1 can increase the progression of steroid-induced osteonecrosis of femoral head,and the expression level is consistent with the effect of biomechanics on steroid-induced osteonecrosis of femoral head;MALAT1/miR-329-5p/PLDL1 signaling pathway may be involved in the development of SIOFH through competitive endogenous RNA mechanisms and the effect of biomechanics on steroid-induced osteonecrosis of femoral head.Part three: In vitro cell experiments are often supplemented by in vivo experiments in scientific research to further verify the conclusion that the change of MALAT1,miR-329-5p,PLDL1 mRNA,and PLDL1 protein in the induced to differentiate after dexamethasone intervention in RAW264.7 and MC3T3E1 were consistent with the result of femoral head tissue in the rat model.After dexamethasone intervention,we we transfected into siMALAT1 to induce cells differentiation,and both MALAT1 and PLDL1 showed reduced expression,while miR-329-5p expression increased significantly,which was intrinsic to non-coding RNA competition.We conclude that MALAT1/miR-329-5p/PLDL1 may increase PLDL1 protein expression in steroid-induced femoral head necrosis,which in turn increases osteoclast differentiation and activity and inhibits osteoblasts differentiation formation and activity,to reduce the bone mass in the femoral head,which eventually leads to the occurrence of osteonecrosis of femoral head.[Conclusions]1.Biomechanical stimulation can accelerate the pathological progress of steroid-induced osteonecrosis of femoral head.Reducing or reducing biomechanical stimulation can delay the progress of its pathological process.Micro-CT scans show that abnormal bone metabolism plays an important role in steroid-induced osteonecrosis of femoral head.2.The results of RT-PCR and Western blotting analysis of femoral head tissue in rats with steroid-induced osteonecrosis of femoral head showed that the MALAT1/miR-329-5p/PLDL1 signaling pathway significantly changed in the occurance of steroid-induced osteonecrosis of femoral head,and biomechanical intervention after SIOFH.3.In vitro experiments,RAW264.7 and MC3T3E1 was transfected siMALAT1 after dexamethasone intervened to analyses changes of signal pathway,MALAT1/miR-329-5p/PLDL1 may participate in the occurrence and progression of steroid-induced osteonecrosis of femoral head through the ceRNA mechanism.